RESULTS Complete or partial remission was noted in 68 of 90 patients (75.5%) after a median follow up of 56 (5–213) months. At follow
up significant median changes were noted in SLE patients: erythrocyte sedimentation rate (ESR) from 44 to 22 mm 1st hour;
anti-dsDNA antibody concentrations from 81 to 48 IU/ml; proteinuria from 2.5 to 1.5 g/day; serum albumin from 36 to 40 g/l;
complement C3 from 0.88 to 0.90 g/l, and C4 from 0.18 to 0.22 g/l. In the vasculitis patients significant median changes were
seen in: ESR from 44 to 15 mm 1st hour; C reactive protein (CRP) from 16 to 5 g/dl; neutrophils from 8.55 to 4.3 × 109/l; platelets from 340 to 261 × 103/l, and haemoglobin from 12.6 to 13.2 g/dl. Patients with Churg-Strauss syndrome, Wegener’s granulomatosis, and neuropsychiatric
lupus showed the best initial response but 58% of Wegener’s patients relapsed. Median corticosteroid doses were significantly
reduced from 15 (5–60) mg to 10 (3–35) mg daily. Adverse events: infections (7 patients), neutropenia (5), lymphopenia (18),
and haemorrhagic cystitis (1 intravenous and 2 oral cyclophosphamide), allergies to mesna (2). None of the women at risk had
prolonged amenorrhoea. Five patients doubled their serum creatinine and five died from sepsis (2) or severe disease (3).

CONCLUSION Treatment of severe connective tissue diseases with ‘low dose’ intravenous cyclophosphamide pulses compares in efficacy with
the higher monthly doses previously advocated. Treatment was well tolerated with fewer adverse effects and most significantly,
there were no cases of premature ovarian failure.

Intravenous pulse cyclophosphamide treatment is widely used in the management of autoimmune connective tissue diseases and
has resulted in a dramatic improvement in patient outcome. Perhaps the most important application has been in the therapeutic
approach to the vasculitides, especially the clinical experience with cyclophosphamide in treating Wegener’s granulomatosis.

Untreated Wegener’s granulomatosis usually has a rapidly fatal course with a mean survival of five months and a one year mortality
of 82%.12 Although corticosteroids may improve the mean survival to 12.5 months, it is clear that the long term prognosis is not significantly
changed by corticosteroids alone.3 The introduction of cyclophosphamide has radically improved survival4 and has also proved to be beneficial in other vasculitic syndromes 5 as well as in active polymyositis.6

Cyclophosphamide is well established in the treatment of lupus nephritis where it reduces the risk of end stage renal failure.7-10 Currently, intravenous doses of cyclophosphamide calculated on a surface area basis have tended to be high (of the order
of 750–1250 mg) and the infection rate has consequently been high—in some series up to 25%. 811

Over the past 10 years, in an attempt to reduce the incidence of infections and other adverse effects, we have treated patients
with severe connective diseases using a less aggressive intravenous cyclophosphamide regimen that does not aim to induce leucopenia
as a treatment goal in the induction phase of treatment. Our reasons were twofold. Firstly, we felt that the lower dose regimen,
being better tolerated was more suitable for outpatient practice. Secondly, we felt that there was an urgent need to attempt
to reduce the three most serious complications of cyclophosphamide: premature ovarian failure, infection (especially Herpes
zoster), and haemorrhagic cystitis in our patients.

We have described our preliminary experience with this regimen.1213 We present here a retrospective analysis of 90 patients with severe connective tissue diseases treated with a total of 883
pulses using this low dose intravenous cyclophosphamide regimen.

Methods

PATIENTS

Ninety patients diagnosed as having severe progressive connective tissue diseases were given low dose intravenous cyclophosphamide
treatment between January 1990 and November 1994 and all were followed up until April 1995. These patients were new referrals
or patients who had previously either relapsed or developed new organ involvement despite treatment with oral prednisolone
(n=38), azathioprine (n=31) or methylprednisolone pulses (n=31), or all three. These patients were followed up in the Lupus
and Vasculitis clinics at St Thomas’s Hospital for a median of 56 (range 5–213) months after the first pulse of cyclophosphamide.
There were 43 patients with systemic lupus erythematosus (SLE), 42 with systemic vasculitides, four with idiopathic inflammatory
myopathies, and one patient with mixed essential cryoglobulinaemic vasculitis. Patients with SLE, Wegener’s granulomatosis,
Churg-Strauss syndrome, and polyarteritis nodosa were classified according to the respective American College of Rheumatology
classification criteria.1415 There were 68 women and 22 men, with a median age of 48 (22–76) and a median disease duration of 94 (18–250) months. Forty
women aged under 40 all had regular menses. There were 71 white, 12 Asian, and seven Afro-Caribbean patients.

CLINICAL ASSESSMENT

Complete clinical examination and laboratory tests were carried out before treatment with intravenous cyclophosphamide, after
the first three to four pulses to assess the initial response to therapy, at the end of the treatment course, and at the end
of the follow up period. All patients were assessed for disease duration, clinical evidence of organ involvement, erthrocyte
sedimentation rate (ESR), C reactive protein (CRP), neutrophil, lymphocyte and platelet counts, haemoglobin value, urine analysis,
24 hour urine protein excretion and creatinine clearance, blood urea, serum creatinine, and albumin. Complement values (C3
and C4) were assayed by radial immunodiffusion (Behring). Anti-ds DNA antibodies were measured by solid phase enzyme linked
immunosorbent assay.16 In terms of assessing ovarian function, we arbitrarily defined premature ovarian failure as prolonged amenorrhoea for at
least 12 months in a woman with previously regular menses under the age of 40.

CRITERIA FOR REMISSION

Complete clinical remission was arbitrarily defined as the complete absence of clinical and laboratory evidence of disease
activity for at least three months. Partial remission was defined as clear evidence of improvement in disease activity with
at least 50% improvement in laboratory parameters for at least three months. Patients who did not meet these criteria were
regarded as treatment failures. Poor renal outcome was defined as a doubling of serum creatinine or the development of end
stage renal failure requiring dialysis.

INDICATIONS FOR INTRAVENOUS CYCLOPHOSPHAMIDE TREATMENT

Patients who received intravenous cyclophosphamide treatment were either new patients receiving no therapy who had active
disseminated systemic vasculitis complicated by renal, pulmonary, cardiac or neurological involvement; SLE complicated by
glomerulonephritis, widespread cutaneous vasculitis, severe cardiac or pulmonary disease, central or peripheral neurological
involvement or patients with these diseases who developed these complications despite treatment with low dose prednisolone
or azathioprine, or both. Intravenous cyclophosphamide was contraindicated in any patient who was pregnant or with evidence
of sepsis. Those with significant renal impairment (creatinine clearance less than 30 ml/min) received 250 mg pulses of cyclophosphamide.
Patients receiving azathioprine had the drug stopped for at least a week before starting intravenous cyclophosphamide to avoid
excessive immunosuppression and bone marrow toxicity.

INTRAVENOUS CYCLOPHOSPHAMIDE

Intravenous cyclophosphamide (500 mg) was given in 100–500 ml saline over one hour accompanied by adequate oral hydration
to promote frequent voiding of dilute urine in the first 24 hours after drug administration. To reduce bladder toxicity further,
mesna (Uromitexan, Boehringer) was given in 816 of the 883 pulses.17 Mesna was initially given (100 mg) intravenously at the start of the cyclophosphamide infusion followed by 200 mg orally
at four and eight hours. Antiemetics (metoclopramide or where necessary ondansetron) were used in all patients. Treatment
was given weekly providing there was no evidence of severe neutropenia at the time of the next intravenous pulse.

Once a clear response (either complete or partial remission) was seen and in particular once major organ involvement was seen
to be improving after weekly intravenous cyclophosphamide treatment, maintenance therapy was started with either azathioprine
(2 mg/kg/day) or continued monthly intravenous cyclophosphamide (500 mg pulses). The choice depended on the clinical response
to the initial pulse cyclophosphamide treatment: azathioprine was used in patients achieving complete remission and monthly
intravenous cyclophosphamide was continued in patients who had either not responded or had achieved partial remission with
continuing disease activity.

STATISTICAL ANALYSIS

Descriptive statistics for the patients were obtained and are reported as medians with ranges or frequencies as appropriate.
Non-parametric tests, Wilcoxon’s rank sum test, and the χ2 test were used in analysis. Statistical significance was indicated by a p value < 0.05.

Results

Seventy four patients initially received weekly intravenous cyclophosphamide therapy: 44 received weekly pulses for a median
of three consecutive weeks (range 2–10) followed by monthly pulses for a median of four (1–42) months and 30 patients received
only weekly intravenous cyclophosphamide pulses for a median of three (2–9) weeks followed by maintenance treatment. Sixteen
patients were given intravenous cyclophosphamide every month for a median of eight (3–37) months.

Most patients (78 of 90, 87%) received 500 mg pulses, though four patients with severe systemic vasculitis who were obese
received 750 mg pulses and eight patients who had renal impairment (creatinine clearance less than 30 ml/min) received 250
mg pulses. The patients were subsequently treated with prednisolone alone (n=48), prednisolone and azathioprine (n=26), oral
cyclophosphamide (n=11), etoposide (n=3) or methotrexate (n=2). Twenty two patients received a second course of monthly pulses
of cyclophosphamide indicated by the clinical severity of the disease or disease relapse. Table 1 shows the median number of pulses and cumulative dose of intravenous cyclophosphamide given according to diagnosis.

Total and median numbers of pulses and cumulative doses of intravenous cyclophosphamide received by patients in each diagnostic
group

RESPONSE TO TREATMENT

Complete or partial disease remission was noted in 68 of 90 patients (75.5%) at the end of the median follow up period of
56 (5–213) months (table 2). The most striking improvements were noted in patients with Churg-Strauss and Wegener’s granulomatosis especially in patients
with upper airway and ocular involvement, usually within two to six weeks of the start of intravenous cyclophosphamide. The
median (range) period to complete remission in those who responded to treatment was: SLE: 21.5 (1–2) weeks, undifferentiated
systemic vasculitis: 15 (4–47) weeks, Wegener’s granulomatosis: 22 (9–175) weeks, and Churg-Strauss syndrome: 22 (21–31) weeks.

Clinical response to induction and maintenance treatment at the end of follow up

Overall, the median prednisolone dose was significantly reduced from 15 (5–60) mg daily at the start of intravenous cyclophosphamide
treatment to 10 (3–35) mg daily (p<0.05) at the end of follow up.

There were 22 of 90 patients (24.5%) who failed to respond to treatment. These included six lupus nephritis patients, two
lupus patients with severe interstitial lung disease and two with cerebral lupus, one patient with dermatomyositis, four patients
with Wegener’s granulomatosis, four with undifferentiated systemic vasculitis, and three patients with polyarteritis nodosa.
Of these patients, histology review showed predominantly chronic or fibrotic changes in renal or muscle samples in six patients.
There were five deaths and six patients were lost to follow up, one with systemic vasculitis and five with SLE.

SYSTEMIC LUPUS ERYTHEMATOSUS

Forty three patients with SLE were treated with intravenous cyclophosphamide with a median of six (2–26) pulses per patient
with a median cumulative dose of 2.5 (1–13) g. In the SLE group as a whole, there were significant improvements after a median
of three (2–7) pulses in ESR, 24 hour protein excretion, serum albumin, anti-dsDNA antibody and C4 values supporting our clinical
finding of a rapid response to weekly intravenous cyclophosphamide (table 3). Maintenance treatment started after the initial weekly infusions resulted in continued significant improvements from baseline
in ESR, 24 hour protein excretion, serum albumin, anti-dsDNA antibody and C3 and C4 values.

Laboratory changes before and after intravenous cyclophosphamide treatment in SLE. Values are median (range)

Twenty six patients had lupus nephritis with persistent proteinuria (>1 g/day) or abnormal urine sediments, or both, confirmed
by renal biopsy in 22 patients. Ten biopsy samples had focal proliferative nephritis (WHO grade III) and 12 showed diffuse
proliferative nephritis (WHO grade IV). Twenty of these 26 patients with lupus nephritis responded well to intravenous cyclophosphamide
treatment. Table 3 shows that there were significant improvements in median 24 hour proteinuria, anti-dsDNA antibody values, serum albumin and
C3 and C4 values. The median serum creatinine increased from 93 (45–250) μmol/l to 112 (51–287) μmol/l at the end of follow
up but the difference was not significant. These patients were given prednisolone alone (n=7), azathioprine (n=11) or oral
cyclophosphamide (n=2).

Fourteen patients with SLE relapsed while receiving treatment: five were receiving weekly followed by monthly treatment, seven
were receiving weekly treatment, and two monthly treatment. These patients were treated with a further weekly course of intravenous
cyclophosphamide therapy (eight patients) or further monthly intravenous cyclophosphamide therapy (six patients). Of these
patients, four went into partial remission and two into complete remission. The other eight patients were considered to be
treatment failures and of these, four patients doubled their serum creatinine concentrations at the end of follow up. Three
of these had diffuse proliferative lupus nephritis (WHO grade IV) and one had focal proliferative lupus nephritis (WHO grade
III). Two of these patients died and are described later.

Ten patients had active cerebral lupus presenting with organic brain syndromes. All had previously received prednisolone treatment
and three received pulse methylprednisolone with no response. Eight patients responded to intravenous cyclophosphamide with
complete resolution of the central nervous system signs and symptoms. One patient, a 17 year old man had two further recurrences
of auditory hallucinations, which again promptly responded to further intravenous cyclophosphamide. Two did not respond one
of whom died and is described below. The other, a 30 year old woman with severe organic brain syndrome who failed to respond
to intravenous treatment improved significantly when given oral cyclophosphamide.

Two patients with interstitial pneumonitis did not respond to treatment and high resolution computed tomography showed predominant
interstitial fibrosis with no evidence of inflammatory ‘ground glass’ changes. Five other patients with SLE complicated by
severe cutaneous and systemic vasculitis all responded to treatment with complete remission.

SYSTEMIC VASCULITIS

Complete or partial remission was seen in 31 of 42 patients (74%) at the end of follow up. The best responses were seen in
patients with Wegener’s granulomatosis where 15 of 19 responded (79%) and three of three patients with Churg-Strauss syndrome
(table 2). However, the median number of pulses received by the Wegener’s granulomatosis patients was higher than any of the other
patient groups (median 11 pulses, range 4–50 with a median cumulative dose of 5.5 g) perhaps reflecting the aggressive relapsing
nature of this disease.

There were significant improvements, within a median of three (2–10) weeks, in the acute phase response as measured by the
ESR, CRP, neutrophil and platelet counts (table 4). This supports our clinical finding of an early and effective response to intravenous cyclophosphamide. Maintenance treatment
resulted in further improvements in these parameters in the group as a whole (table4).

Laboratory changes before and after intravenous cyclophoshamide treatment in systemic vasculitis. Values are median (range)

Although most patients with Wegener’s granulomatosis responded initially, there was a significant relapse rate. Of the 19
patients with Wegener’s granulomatosis, 11 (58%) relapsed a median of 8.6 months after induction therapy. Seven of these patients
who relapsed had received weekly followed by monthly intravenous cyclophosphamide treatment (five patients) or weekly treatment
(two patients). The other four patients relapsed while receiving maintenance treatment. Four patients received further weekly
intravenous cyclophosphamide treatment and three received further monthly treatment and of these there was only one further
relapse, which again responded to weekly treatment.

In all, 10 of 11 patients who relapsed responded well to further weekly intravenous cyclophosphamide, the other patient being
switched successfully to etopside. The most notable finding in the systemic vasculitis group was that 13 patients, who did
not have renal disease, were successfully maintained with azathioprine after intravenous cyclophosphamide treatment.

Four patients with Wegener’s granulomatosis did not respond to intravenous cyclophosphamide. One patient was switched to oral
cyclophosphamide and three others received monthly oral etoposide with good results one of whom has previously been reported.18One Wegener’s granulomatosis patient doubled his serum creatinine by the end of follow up. Two patients died and are described
below.

ADVERSE EFFECTS

A total of seven of 90 patients (7.8%) had serious infections during the entire follow up period. These included three episodes
of Herpes zoster (one intravenous and two oral cyclophosphamide), three episodes of pneumonia (one intravenous and two oral
cyclophosphamide), three episodes of septicaemia (one intravenous and one oral cyclophosphamide, one azathioprine). One patient
receiving oral cyclophosphamide had mild alopecia. Three episodes of haemorrhagic cystitis (one intravenous and two oral cyclophosphamide)
and five episodes of neutropenia were seen (one intravenous and two oral cyclophosphamide and two azathioprine). Two patients
had allergic skin reactions to the mesna. Overall, in the patients receiving intravenous cyclophosphamide, there was no correlation
between the number of pulses, the cumulative cyclophosphamide dose, and the incidence of infection. One patient with lupus
nephritis did however have two infections after receiving two courses of intravenous cyclophosphamide and a total of 26 pulses.
There was a non-significant trend to infection in patients receiving either daily oral cyclophosphamide (n=11) or etoposide
(n=3) maintenance treatment (p=0.065, odds ratio 3.7 (0.97–14.5)). No malignancies were noted.

Tables 3 and 4 show that neutrophil, lymphocyte, and platelet counts were marginally reduced during the course of follow up. No correlation
was found between the number of pulses, cumulative doses of cyclophosphamide and neutropenia or lymphopenia, or both.

Among the 68 women, 28 were known to have been perimenopausal or postmenopausal at the start of intravenous cyclophosphamide
treatment and 40 had regular menstruation. No premenopausal woman who received intravenous cyclophosphamide suffered premature
ovarian failure. Table1 shows that the median cumulative dose received by patients with SLE who were most at risk of ovarian failure was only 2.5
g. Of the 11 patients who received oral cyclophosphamide, five were male and six female: two postmenopausal, four with regular
menstruation. All four of the non-menopausal women suffered prolonged amenorrhoea with oral cyclophosphamide.

There were five deaths in all, three with SLE and two with Wegener’s granulomatosis. A 30 year old woman with lupus nephritis
had severe persistent neutropenia that may have been related to both disease and treatment—she developed Gram negative septicaemia
and died after 10 months of follow up. A 41 year old woman with lupus nephritis died after eight months of follow up from
a severe flare of her disease with renal and central nervous system disease that failed to respond to treatment. A 51 year
old man with lupus psychosis died from severe SLE that failed to respond to 12 pulses of intravenous cyclophosphamide. Two
patients with Wegener’s granulomatosis died: one from a pneumonia associated with treatment and one from overwhelming disease
that failed to respond.

Discussion

Cyclophosphamide is a potent immunosuppressive agent acting on T and B lymphocytes.1920 While extremely effective, its use is associated with serious adverse effects including bacterial infections, Herpes zoster,
haemorrhagic cystitis, neoplasias including bladder malignancies, and premature ovarian failure.52122 To minimise these drawbacks, we have introduced the use of cyclophosphamide in an intermittent intravenous low dose pulse
regimen.

We studied the long term effect of this regimen in the induction phase of treatment in 90 patients with severe connective
tissue diseases having followed them up for a median of 56 months. An excellent response in both clinical and laboratory parameters
was seen after the first four weeks of treatment, which was sustained with subsequent maintenance treatment. The clearest
long term response to our therapeutic regimen was seen in patients with Churg-Strauss syndrome, neuropsychiatric lupus and
in most patients with lupus nephritis. Although there was a high relapse rate in the Wegener’s granulomatosis patients, all
but one promptly responded to further intravenous cyclophosphamide treatment.

The previously published regimens of intravenous cyclophosphamide, often requiring doses in excess of 1 or 1.5 g have been
associated with significant adverse effects, with infections in up to 10% and Herpes zoster in as many as 25%.822 Sustained amenorrhoea has been seen in up to 39% of patients receiving a prolonged monthly treatment with a high dose intravenous
cyclophosphamide regimen (a total of 12 pulses or more).23

Infectious complications were seen in only seven patients (7.8%), though two of these patients died of sepsis associated with
treatment. Herpes zoster was seen in only one patient (1%) receiving intravenous cyclophosphamide compared with 25% in previous
series.822 The neutropenia associated with intravenous cyclophosphamide was mild and transient in four of five patients, the fifth patient
suffering a fatal infection as described above. Twenty patients were lymphopenic before pulse cyclophosphamide and 18 patients
had a further reduction of the lymphocyte count during the treatment course. At the end of the study, this reduction was not
statistically significant. We did not observe any significant episodes of thrombocytopenia associated with low dose cyclophosphamide
pulses in any patient.

None of our patients suffered ovarian failure associated with intravenous cyclophosphamide. This may be related to the relatively
low cumulative dose of intravenous cyclophosphamide—indeed, prolonged menstrual irregularities were only noted in patients
receiving long term oral cyclophosphamide. However, premature prolonged amenorrhoea can occasionally occur long after the
initial exposure to cyclophosphamide and we continue to monitor our patients for this, particularly given the consequences
for osteoporosis.

Mesna was given in 816 of 883 pulses. We had only one patient who suffered from haemor- rhagic cystitis during the course
of the study. Perhaps significantly, this occurred in one of the patients in whom mesna was omitted. We did not observe any
episodes of bladder cancer and these patients continue to be followed up closely. Maculopapular drug eruptions were seen in
two patients associated with mesna, a com- plication previously reported by Zonzitset al.24

In terms of efficacy, 22 patients (24.4%) were considered to be ‘treatment failures’ at follow up. Four lupus nephritis patients
and one Wegener’s granulomatosis patient had more than doubled their serum creatinine concentrations at the end of the study.
The risk of doubling serum creatinine was higher among patients with an initial serum creatinine greater than 150 μmol/l (three
of five patients). In comparison, Boumpas et al25 noted a 35% failure rate in patients treated with ‘short-course’ monthly intravenous cyclophophamide (35% doubled serum creatinine
including 25% with end stage renal failure). Similarly, Belmont et al26 noted that 30% of patients failed to maintain stable renal function using monthly intravenous pulses of cyclophosphamide
after a mean (SD) of 52 (3) months of follow up. Clearly though direct comparisons between these studies is difficult particularly
as our regimen incorporated long term azathioprine as maintenance. Furthermore long term follow up is essential to observe
differences that can develop much later.10

Although the median prednisolone dose at the end of follow up was 10 mg daily, the range was wide and included patients who
still had some disease activity at the time of follow up. The initial corticosteroid dose was relatively low considering the
severity of disease and it is possible that oral prednisolone may need to be given for longer periods given the lower doses
of cyclophosphamide used in this regimen. This has obvious implications for side effects and osteoporosis prophylaxis in particular
is now considered in all these patients.

The main limitations of this study include its retrospective nature with all the problems that entails, including the lack
of a standard lupus disease activity measure, which should ideally be used in a prospective way. The patients are very heterogenous,
reflecting clinical practice and different maintenance treatments have been used, perhaps highlighting our uncertainty about
the ideal treatment for keeping these diseases under control once remission has been achieved. Nevertheless, our results are
encouraging enough to embark upon a randomised prospective study of the low dose intravenous cyclophosphamide regimen in comparison
with the standard high dose monthly intravenous cyclophosphamide regimen in the treatment of proliferative lupus nephritis
that is currently under way in Europe.

In summary, our clinical experience with this large cohort of patients suggests that the low dose intravenous cyclophosphamide
regimen was as therapeutically effective as other previously published regimens with far fewer adverse effects and good tolerance,
important in outpatient clinic practice. The most important advantage of this regimen seems to be the relative lack of ovarian
toxicity.